Mammalian aging is associated with functional defects in numerous tissues that require cellular renewal during life. These tissues are replenished by tissue stem cells as differentiated cells are lost throughout life. Accumulating evidence indicates that defects in aging tissues can be explained in part by impaired stem cell function or alterations in the stem cell niche. A greater understanding of how stem cell regulation is altered in aging is required in order to devise approaches to improve or reverse defects in renewing tissues with advancing age. We have found that two pathways with critical roles in stem cell survival and self-renewal - telomerase and Wnt signaling - are intimately connected. Synthesis of telomeres by telomerase is required for stem cell self-renewal. In addition, telomerase serves noncanonical functions in supporting stem cell function independent of its role in telomere elongation. To complement our original Aims in Project 2, we have made a major investment in the development of new mouse genetic tools that enable us to detect telomerase-positive cells in vivo for the first time. We will use these new technologies to determine which cells in the intestine, and in the skin and its appendages, express telomerase. We will determine the relationships between these telomerase-positive cells and previously identified stem cell populations. We will assess cell cycle in telomerase-positive cells to understand whether telomerase is associated with prolfierating stem cells or quiescent stem cells. We will characterize telomerase-positive cells by RNA-seq and in functional assays. These studies will greatly support our work in understanding non-canonical telomerase functions in vivo.